The .beta.-lactamases are enzymes produced by many pathogenic microorganisms such as bacteria of the genera Staphylococcus, Escherichia, Klebsiella, Proteus, Pseudomonas, Enterobacter, etc. They have the capacity to open the .beta.-lactam ring of antibacterial agents with this structural feature, notably the penicillins and cephalosporins. This reaction inactivates the effectiveness of the antibacterial agent in combatting infection caused by the bacteria which in turn gives rise to resistance by strains of the disease-causing microorganism to treatment with the antibacterial agent. A .beta.-lactamase inhibitor can therefore be useful in protecting the antibacterial agent against degradation by the enzyme and thus enhance the effectiveness of the antibacterial agent.
Cultures from Actinomycetes have been reported as producing .beta.-lactamase inhibiting substances. See British Pat. No. 1,363,075; J. Antibiotics 25, 473 (1972), 26, 51 (1973).
Species of Micromonospora have been described which produce antibiotics such as gentamicin, sisomycin, neomycin and others. See, for example, J. Antibiotics 27, 493 (1974); U.S. Pat. No. 4,078,056, Mar. 7, 1978. These Micromonospora produced antibiotics have typical spectra of antibacterial activity against various microorganisms such as Streptococcus fecalis, Staphylococcus aureus, Escherichia coli, Candida albicans, etc., but the production of .beta.-lactamase inhibitors by this genus has not been reported.
We have now found that species of the microorganism Micromonospora can be made to produce the substance EM4615 which inhibits the effect of .beta.-lactamase enzymes. EM4615 is produced by culturing species of the microorganism Micromonospora at about 20.degree. to 35.degree. C., preferably about 25.degree. under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable carbohydrate and nitrogen source for about 48 to 240 hours, preferably about 144 hours. The .beta.-lactamase inhibitor EM4615 can then be extracted from the fermentation broth. It is isolated by centrifuging the completed broth to remove the mycelium and other solids, extracting the remaining broth with a lower alkanol, preferably n-butanol, at a low pH, e.g., approximately pH 2, then back extracting the alcohol extract with water at about pH 7 to 9. The water extract contains the .beta.-lactamase inhibitor.
This invention therefore relates to the .beta.-lactamase inhibitor EM4615 and to a method for producing it.